Matrix metalloproteinase-9 undergoes expression and activation during dendritic remodeling in adult hippocampus

Abstract

Neurons of adult brain are able to remodel their synaptic connections in response to various stimuli. Modifications of the peridendritic environment, including the extracellular matrix, are likely to play a role during synapse remodeling. Proteolytic disassembly of ECM is a complex process using the regulated actions of specific extracellular proteinases. One of best-characterized families of matrix-modifying enzymes is the matrix metalloproteinase (MMP) family. Here, we describe changes in the expression and function of two well known MMPs, MMP-9 and MMP-2, in adult rat brain before and after systemic administration of the glutamate receptor agonist kainate. Kainate application results in enhanced synaptic transmission and seizures followed by selective tissue remodeling, primarily in hippocampal dentate gyrus. MMP-9 but not MMP-2 was highly expressed by neurons in normal adult rat brain. MMP-9 protein was localized in neuronal cell bodies and dendrites. Kainate upregulated the level of MMP-9 mRNA and protein within hours after drug administration. This was followed several hours later by MMP-9 enzymatic activation. Within hippocampus, MMP-9 mRNA and activity were increased selectively in dentate gyrus, including its dendritic layer. In addition, MMP-9 mRNA levels decreased in areas undergoing neuronal cell loss. This unique spatiotemporal pattern of MMP-9 expression suggests its involvement in activity-dependent remodeling of dendritic architecture with possible effects on synaptic physiology.

Fig. 1.

Tissue distribution of MMP-9 and MMP-2 in adult rat brain. Sections of dorsal hippocampus demonstrating the distribution of MMP-9 and MMP-2 mRNA and protein are shown.A, Nissl staining of a hippocampal coronal section.mol. layer, Molecular layer; lacun. mol., lacunosum molecular layer; rad., radiatum layer. Scale bar, 1 mm. B, Tissue distribution of MMP-9 mRNA (in situ hybridization; left image) and protein (immunohistochemistry; right image). MMP-9 mRNA is enriched in neuronal fields of the hippocampus. The right image shows enrichment of MMP-9 protein in neuronal bodies of the dentate gyrus and the CA subfields. Diffuse MMP-9 protein staining, especially in the lacunosum molecular layer, can also be discerned.C, Tissue distribution of MMP-2 mRNA (left image) and protein (right image). In contrast to MMP-9, MMP-2 mRNA and protein are diffusely distributed over the entire hippocampus.

Fig. 2.

Cellular localization of MMP-9 and MMP-2 protein in adult rat brain. Colocalization of MMPs with cell type-specific markers is shown. A, DG staining with MMP-9 (left image) and the neuronal maker NeuN (middle image). Note NeuN-positive granule neurons that are MMP-9 positive. Cell nuclei are labeled with DAPI (right image). B, DG staining with MMP-9 (left image) and the astrocyte marker GFAP (middle image). Arrows indicate astrocytes that express MMP-9 protein. Cell nuclei are labeled with DAPI (right image). C, Neocortex staining with MMP-9 (left image) and the dendritic marker MAP-2 (middle image). Arrows indicate MMP-9-positive dendrites. Cell nuclei are labeled with DAPI (right image). D, DG staining with MMP-2 (left image) and GFAP (middle image). Note GFAP-expressing astrocytes that are MMP-2 positive. Cell nuclei are labeled with DAPI (right image). Scale bar, 50 μm.

Fig. 3.

MMP-9 is localized in neuronal processes of cultured hippocampal neurons. Co-immunostaining of dissociated hippocampal neurons in culture with anti-MMP-9 antibody Ab.1 and anti-TUJ1 antibody is shown. Although dissociated neurons did not show clear neuritic expression of the enzyme at low magnification (top images; scale bar, 500 μm), when the cells were analyzed at high magnification, MMP-9 protein was localized clearly in TUJ1-positive neuronal processes (bottom images; scale bar, 50 μm).

Fig. 4.

Characterization of MMP activities from rat brain.A, Gel zymography of brain extracts (see details in Materials and Methods). Left image, Triton X-100-soluble (lanes 1, 3, 5) and -insoluble (lanes 2, 4, 6) fractions from neocortex (N-CX), hippocampus (HIP), and limbic cortex (L-CX) show a similar molecular pattern of enzymatic activities of MMPs. Two main activities at ∼97 kDa (bands a, b) and one at 70 kDa (band c) can be detected. Note the enrichment of MMPs in the Triton X-100-insoluble fraction. Immunoprecipitation with the selective anti-MMP-2 (lane 7) or anti-MMP-9 (lane 8) antibody confirms that the 97 kDa doublet consists of latent (band a) and active (band b) forms of MMP-9 (lane 8), whereas the 70 kDa activity is MMP-2 (line 7). Right image, One hundred five kilodalton recombinant mouse proMMP-9 (latent MMP-9; lane 9) and 70 kDa recombinant human proMMP-2 (latent MMP-2;lane 10) were co-electrophoresed with MMPs extracted from rat hippocampus (lane 11). Mouse MMP-9 migrates slightly above extracted rat 97 kDa gelatinase, whereas MMP-2 migrates directly at the level of extracted 70 kDa gelatinase. The discrepancy in MMP-9 migration is related to differences in both protein size and level of glycosylation. B, Western blot of latent (proMMP-9) and active forms of human recombinant MMP-9 detected with the anti-rat-MMP-9 antibody. Note that the anti-rat-MMP-9 antibody cross-reacts with human MMP-9.

Fig. 5.

Kainate upregulates MMP-9 protein and induces its enzymatic activation. A, Representative sets of zymograms of either Triton X-100-soluble or -insoluble fractions from rat hippocampi after kainate treatment. Activities of latent (band a) and active (band b) MMP-9 are upregulated after 6 and 24 hr, respectively. The activity of MMP-2 remains unchanged (band c). B, Zymographic analysis of MMP-9 immunoprecipitated with the selective anti-MMP-9 antibody (Ab.1) from hippocampal protein extracts obtained from kainate-treated rats (from the same animals as analyzed inA). Activities of latent (band a) and active (band b) MMP-9 are upregulated after 6 and 24 hr, respectively. C, MMP-9 Western blot analysis of Triton X-100-insoluble fractions from hippocampal protein extracts after kainate treatment (from the same animals analyzed in A). Samples from Triton X-100-insoluble fractions at different time points (0, 2, 6, and 24 hr) were subjected to Western blotting with the anti-MMP-9 antibody. Latent (band a) and active (band b) forms of MMP-9 are upregulated after 6 and 24 hr, respectively.

Fig. 6.

In situ hybridization detection of MMP-9 and MMP-2 mRNAs after kainate treatment. Rat brain sections subjected to either Nissl staining (middle images) orin situ hybridization with MMP-9 (left images) or MMP-2 (right images) radiolabeled cDNA probes. Sections were made from either control (contr.) animals (A, E, I) or kainate-treated animals at 6, 24, and 72 hr after drug administration (B–L). Kainate treatment causes neurodegeneration and significant tissue deterioration after 24 and 72 hr in piriform cortex and amygdala as visualized by the decrease in intensity of the Nissl staining (G, H). Kainate treatment results in upregulation of MMP-9 mRNA in DG (B–D) and downregulation in degenerating limbic areas (C, D). MMP-2 mRNA was downregulated in lesioned limbic areas (K, L).

Fig. 7.

Kainate induces redistribution of MMP-9 message in the dentate gyrus. In situ hybridization detection of MMP-9 mRNA in rat hippocampi, control (contr., top images) and 24 hr after kainate-induced seizures (two representative brains, A, B) is shown. Rat brain sections were subjected to either in situ hybridization with the MMP-9-radiolabeled probe (left images) or Nissl staining (right images). Note enhancement and relocation of MMP-9 mRNA from the granular layer (GL) to the molecular layer (ML) of the dentate gyrus.

Fig. 8.

Increase of MMP-9 mRNA in the DG detected by RT-PCR after kainate treatment. The DG was removed from 1-mm-thick hippocampal slices and subjected to total cellular RNA extraction. RT-PCR analysis demonstrated that DG MMP-9 mRNA was upregulated 24 hr after kainate treatment. No changes were seen for MMP-2 and β-actin mRNAs. contr., Control.

Fig. 9.

MMP-9 immunostaining in kainate-treated brains. Representative images show MMP-9 protein expression in hippocampus at different time points after kainate treatment. Thearrowhead indicates enhancement of diffuse staining in the lacunosum molecular layer. The arrow indicates the border between the molecular layer (ML) of the DG and the lacunosum molecular layer of CA1. MMP-9 protein was upregulated in the granular layer 6 hr after the treatment. Seventy-two hours after kainate treatment, diffuse staining of MMP-9 was enhanced in the DG, whereas in the CA subfields, MMP-9 was downregulated. Note that kainate produced neurodegeneration resulting in partial deterioration of the hippocampus (CA3 and CA1 fields). contr., Control.

Fig. 10.

Cellular localization of MMP-9 protein in kainate-treated brains. Colocalization of MMP-9 protein with cell type-specific markers is shown. In control (contr., left images) and 24 hr after kainate application (right images), DG staining with MMP-9 (top images) and the astrocyte marker GFAP (middle images) is shown. Sections from the same animals were stained with the neuronal marker NeuN (bottom images). MMP-9 immunostaining was enhanced in the granular layer (GL) and molecular layer (ML). H, Hilus. Scale bar, 120 μm.

Fig. 11.

In situ zymography detects gelatinase activity in hippocampal sections. Brain sections were incubated with different fluorescent substrates: gelatin (gelatinase substrate; A, C, D) and casein (panproteinase substrate;B). Cleavage of the substrate by a proteinase results in unblocking of quenched fluorescence and an increase in fluorescence. Note that the pattern of gelatin cleavage resembles MMP-9 distribution (A). Gelatinase (gel.) activity is attenuated by the zinc chelator 1,10-O-phenantroline (Phen., C) and the synthetic MMP inhibitor (MMPI, D; see details in Materials and Methods). Scale bar, 1 mm.

Fig. 12.

Kainate upregulates gelatinase activity in granular and molecular layers of the hippocampus. A, Brain sections from control (contr.) and kainate-treated (KA; after 24 hr) brains were analyzed by means ofin situ zymography. The optical density of the granular layer (GL) and molecular layer (ML) were measured by NIH Image software (graphs). Note, that the kainate treatment enhances gelatinase activity in both granular and molecular layers of the hippocampus (differences between control and kainate-treated brains are statistically significant byt test; p < 0.01;n = 5; error bars represents SEM).B, Control and kainate-treated brains (24 hr;n = 2) were cryocut into 60 μm sections. The upper molecular layer (mol. layer, ML) of the DG was dissected from sections under the microscope, collected in lysis buffer, and subjected to immunoprecipitation with anti-MMP-9 Ab.1.Left image, Nissl-stained hippocampus from which the molecular layer was dissected. Right image, Upregulation of MMP-9 activity within the molecular layer after kainate treatment.